1. Using heat as a flight stimulant, we found an increase in intensity by density which was significant by ANOVA F test at the
0.05 significance level as tested at frequencies 600Hz, 700Hz (Table 2), and 800Hz (Figure 3). For each decibel increase
in intensity, density increased by 5.67 mosquitoes (2.65-8.69; p = 0.003).
During the heat assay, we also observed a shift in the expected frequency of flight sound from a peak near 400Hz to a
peak near 700Hz.
Development of a novel assay to measure flight capacity of Anopheles gambiae s.l.
Amy R Krystosik1, Diana L. Huestis2, and Tovi Lehmann2
1College of Public Health, Kent State University, Kent, OH
2Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD
Background
Results and Discussion
Acknowledgments
Materials & Methods
Abstract
Conclusions
Future Directions
Monica Artis, BA, Andre Laughinghouse, Kevin Lee, and Kofi Adoo
My mentor Dr Mark James, Ph.D., Kent State University
Guitar Center staff sound training
 Fine tune the activity meter
 Develop flight to exhaustion assay
As this project was exploratory in nature and the primary objective was to develop a protocol and/or assay that would be
piloted in the lab and adapted to the field, there were many assays which did not produce desired results. However, these
methods might be useful in developing a flight to exhaustion assay (Table 1).
This activity meter is useful to measure flight activity in lab reared mosquitos
under controlled conditions. This method could be adapted to field conditions
with minimal effort as the equipment and methods are readily available at the
associated field site in Mali.
Sources Cited
Adamou, A., A. Dao, S. Timbine, Y. Kassogue, A. S. Yaro, M. Diallo, S. F.
Traore, D. L. Huestis and T. Lehmann (2011). The contribution of aestivating
mosquitoes to the persistence of Anopheles gambiae in the Sahel. Malar J.
England. 10: 151.
 Huestis, D. L., A. S. Yaro, A. I. Traore, K. L. Dieter, J. I. Nwagbara, A. C.
Bowie, A. Adamou, Y. Kassogue, M. Diallo, S. Timbine, A. Dao and T.
Lehmann (2012). Seasonal variation in metabolic rate, flight activity and body
size of Anopheles gambiae in the Sahel. J Exp Biol. England. 215: 2013-
2021.
 Kiszewski, A., A. Mellinger, A. Spielman, P. Malaney, S. E. Sachs and J.
Sachs (2004). "A global index representing the stability of malaria
transmission." Am J Trop Med Hyg 70(5): 486-498.
 WHO (2013). WHO | World Malaria Report 2012. WHO. Switzerland, World
Health Organization Global Malaria Programme: 195.
 Sinka, Marianne E, et al. "A Global Map of Dominant Malaria Vectors."
Parasites & Vectors 5.1 (2012): 69. Print.
Anophelines are important vector species in sub-Saharan Africa and
contribute to the continued transmission and burden of malaria worldwide.
The dry-season ecology of anophelines, specifically in the arid Sahel
conditions, remains unknown, but two hypothesis have been proposed to
explain the repopulation phenomenon after the dry season: aestivation and
migration. To investigate the migration hypothesis, we developed an activity
meter to measure flight by sound accounting for environmental conditions.
We found that intensity of sound can predict flight density at frequency of
400-800 Hz; however, this was only achievable at temperatures greater than
63 C in the G3 colony.
A second stimulant used to induce flight was patchouli; but, due to
background noise in the lab, we could not detect change in intensity by cage
density although relative observed flight did increase with cage density.
Further work can expand this activity meter to a flight to exhaustion assay
which is currently under development. These methods may be field-
adaptable, allowing us to study if is it possible that mosquitoes repopulate by
migration.
_cons 49.67919 38.56974 1.29 0.200 -26.63763 125.996
hz400 -.5712355 .7724086 -0.74 0.461 -2.099578 .9571069
flight_level_obs 31.41629 5.063295 6.20 0.000 21.3977 41.43489
density Coef. Std. Err. t P>|t| [95% Conf. Interval]
Total 493396.947 130 3795.36113 Root MSE = 54.423
Adj R-squared = 0.2196
Residual 379117.698 128 2961.85702 R-squared = 0.2316
Model 114279.248 2 57139.6242 Prob > F = 0.0000
F( 2, 128) = 19.29
Source SS df MS Number of obs = 131
_cons 446.292 81.45404 5.48 0.001 258.4587 634.1253
hz700 5.665994 1.309523 4.33 0.003 2.646227 8.68576
Density Coef. Std. Err. t P>|t| [95% Conf. Interval]
Total 45382.4 9 5042.48889 Root MSE = 41.212
Adj R-squared = 0.6632
Residual 13587.1107 8 1698.38883 R-squared = 0.7006
Model 31795.2893 1 31795.2893 Prob > F = 0.0025
F( 1, 8) = 18.72
Source SS df MS Number of obs = 10
The WHO 2012 World Malaria Report estimates the malaria burden in 2010
to have been approximately 219,000,000 cases and 660,000 deaths, of
which 174,000,000 cases and 596,000 deaths were in the African region
(WHO, 2013).
Anopheles gambiae s.l. is an important vector of malaria in sub Saharan
Africa (Kiszewski et al., 2004; Figure 1). However, the dry season ecology of
this species is not well-described (Adamou et al., 2011).
Possible hypotheses for the repopulation after the dry season are aestivation
and/or migration (Adamou et al., 2011, Huestis et al., 2012). In order to test
the hypothesis of migration for repopulation, flight capacity is an important
characteristic. For this reason we are developing an activity index to
determine flight by sound, specifically the characteristics of flight sound
including intensity and frequency.
Using Audacity 2.03 we removed the background noise where noise profiles were collected by environment without
mosquitoes before flight recordings. Flight sound was analyzed by spectrograms of average intensity by frequency
(Figure 2).
Using STATA 12, we made measures of correlation between mosquito density and intensity by frequency. We also used
ANOVAs with density as the independent variable controlling for flight frequency in Hz, intensity in decibels, observed flight
activity, microphone placement and type, environment including room, fan, and stimulus including heat, and patchouli
(Table 1).
Figure 2: Spectrogram of mosquito flight audio
Materials & Methods
We observed an increase in relative flight activity by cage density using
patchouli as a stimulant. However, due to background noise in the insectary
and the larger cage utilized, we do not observe an increase in intensity by
density (Figure 4). There is a difference in flight level by density but not
intensity (Table 3).
There was no significant interaction between flight level observed and
frequency . Also, perhaps due to insufficient washout period between
treatments, we did not observe significant difference in flight activity between
patchouli and non-patchouli treatments.
Results and Discussion
Figure1:Malariavectordistribution(Sinka,2012)
Figure 3: Heat assay plot of Intensity versus Density by Frequency
Table 2: ANOVA density versus intensity by frequency
Figure 4: Intensity versus cage density by relative observed flight level
Table 3: ANOVA intensity versus density by relative observed flight level
Method of
stimulation Results Possible reasons
Wind ineffective Did not prompt long bouts of flight; the noise interfered with recording of flight
Pinning ineffective did not sustain flight; method not suitable for larger cage densities
Tapping effective produced flight but required continuous tapping interfering with mosquito flight recording
Heat effective stimulated mosquito flight at ranges above 36⁰C
Patchouli effective stimulated flight as an irritant especially when combined with cage fans
Table 1: Methods of flight stimulation
The experiment was conducted with the following equipment:
Anopheles gambiae s.l. from the CDC G3 colony
Olympus recorders and microphones
Custom and standard cages modified with sound and heat insulating
materials
In order to produce flight in the mosquitoes, the following methods of
stimulation were used: wind, pinning, tapping, heat, patchouli.